A multi-faceted research project is directed aimed at computational studies of enzymatic processes in aqueous solution. The theoretical approach centers on molecular dynamics free energy simulations of enzymatic reactions using combined quantum mechanical and molecular mechanical (QM/MM methods. To achieve greater accuracy and capability, we propose to further improve the mixed molecular orbital and valence bond (MOVB) theory such that it can be conveniently calibrated, validated and used by biochemists as a computational tool to help interpret experiment findings. The MOVB theory will be implemented and distributed with the capability of using ab initio and semiempirical molecular orbital and density functional theory. In addition, we plan to incorporate explicit polarization effects into combined QM/MM calculations, which can significantly increase the accuracy to describe enzyme and substrate interactions consistently. A major thrust is to provide a deeper understanding of the underlying principles and mechanisms of enzymatic reactions. During this grant period, we aim to elucidate the origin of catalysis in histone lysine demethylases, which catalyze the same chemical transformation by different mechanisms and using different enzyme cofactors. On the other hand, studies of the L-dopa decarboxylase, an enzyme related to the treatment of Parkinson's disease, can provide insight into chemical selectivity by different enzymes employing the pyridoxal phosphate cofactor. Histone lysine demethylases are two classes of enzymes recently discovered to dynamically control the methylation states of chromatin, which are related to gene activation and gene silencing and these enzymes are potential targets for anticancer drugs. In addition, we seek to address the effects of protein dynamics and enzyme reorganization energies on catalysis. The MOVB method provides an important research tool to study these questions, and the results will be of general importance to protein engineering and inhibitor design. PUBLIC HEALTH RELEVANCE: Proteins are workhorses in the living cell, performing all the fundamental tasks from metabolism to cell growth. An important goal is to develop pharmaceutical drugs against protein targets that are responsible for cancer growth and other diseases. The research described in this proposal aims at the fundamental understanding of the mechanism and function of enzymes, proteins that catalyze chemical reactions, and the knowledge gained from these studies can help design inhibitors and engineer specialized proteins for biomedical and industrial applications.